Sunday, June 28, 2015

A decade ago, I began my PhD at
Vanderbilt University in Nashville, Tennessee, where I was interested in
studying the evolutionary process of speciation (or how new biological species
evolve). I was very lucky during my PhD to be surrounded by great people.
Case in point, I shared an office for part of the year with a visiting
collaborator, Patrik Nosil, who studied speciation in a group of stick insects
called Timema. Second, my PhD advisor
encouraged me to invite great thinkers on speciation to be part of my
dissertation committee – enter Jeff Feder from the University of Notre Dame,
who studied speciation in a group of fruit-feeding flies called Rhagoletis and served as my external
committee member. These connections made during the beginning of my PhD last to
this day.

Figure 1. The Pancake Pantry in Nashville, TN, USA.

During a fateful visit to a common grad
student hangout (circa 2007), the Pancake Pantry (Fig. 1), Patrik Nosil and I and a group of graduate students
started discussing the age-old debate about the number of genes involved in
adaptation (and speciation): few versus many? And whether the traits
responsible for adaptation and speciation were polygenic traits or traits with
a simple genetic basis? One way we thought to test this was to use as many
molecular markers as you could survey, distributed across the genome, and ask
the question: how many of these gene regions exhibit significant population differentiation,
but are restricted to populations adapting to different environments? We came
up with ideas of how to test it, and what type of tools we would need, right
over our plates of pancakes! I think we even had a budget by the time we walked
back in our calorie coma from lunch. My
major takeaway from this lunch was that I now considered the genome as an
active player, not a passive mediator, in the speciation process and I would
never think about speciation in the same way again!

What emerged initially from this pursuit were two comparative AFLP genome scans of two different study systems, each
undergoing speciation driven by divergent ecology, that were published in the
journal Evolution (Nosil et al. 2008; Egan et al. 2008). These studies were very informative in
highlighting the proportion of gene regions (AFLPs) in the genome exhibiting strong
differentiation between divergent populations, and possibly addressed the
repeatability of gene regions associated with adaptation to two environments
(in our case, host plants).But we were
also left with many more questions than answers. How were these divergent loci
distributed and arrayed across the genome? And were the loci exhibiting strong differentiation
driven by selection or other evolutionary phenomena?

Fast-forward to 2010 – I finished my PhD and
I was awarded a Faculty Fellowship at the University of Notre Dame, which came
with some seed money for research and the chance to work more closely with my
external committee member, Jeff Feder. Almost immediately upon arriving in
South Bend, IN, Patrik (now in Sheffield, UK), Jeff, and I had a set of
conference calls and email exchanges that started the project that would result
in the Ecology Letters MS I will summarize below. (Jeff and Patrik had just
finished a sabbatical in Berlin the year before where they spent much of their
time ruminating on the genome-level phenomena influencing the speciation
process.) We recruited other evolutionary biologists well trained in Rhagoletis biology (Tom Powell, Glen
Hood, and Greg Ragland), as well as two computer scientists (Scott Emrich and
his PhD student Lauren Assour) with the ability to process the large amount of
data we would gather.

Our interests were to better understand
the role the genome might play in the evolution of new species. We were
inspired by a paper published over 30 years ago by Joe Felsenstein (1981),
where he described the difficulty of building up many-locus differences between
populations if gene flow was ongoing and recombination was breaking up
associations. This conflict between selection and gene flow would form the
basis for our project. How is it that populations can diverge in the face of
ongoing gene flow? What are the properties or characteristics of species that
are suspected of speciation-with-gene-flow which facilitated their divergence?

Rhagoletis
pomonella offered a
great study system to test these ideas, as it is a well-documented case of
speciation-with-gene-flow (Fig. 2). Rhagoletis
pomonella is a member of a sibling species complex containing numerous geographically
overlapping taxa proposed to have radiated in sympatry by adapting to many new
host plants from several different plant families. Rhagoletis flies infest the fruits of their host plants, where host
fruits are typically available for a discrete window of time over the growing
season and each fly species completes one generation per year. Adult flies meet
exclusively on or near the host fruits to mate; females oviposit into the host fruit;
larvae consume the fruit, then burrow into the soil to pupate, entering a
pupal diapause that lasts until the following year. Thus, phenological matching
of fly to host-plant fruiting is critical to fly fitness.

The most recent example of a host shift
driving speciation is the shift of R.
pomonella from its native host hawthorn to introduced, domesticated apple,
which occurred in the mid-1800’s in the eastern United States. Genetic and
field studies have shown that apple and hawthorn flies represent partially
reproductively isolated host races and that gene flow has been continuous
between the fly races since their origin. One key trait that differs between
the races is the timing of diapause termination, which varies between the races
to match the 3–4 week earlier fruiting time of apple versus hawthorn trees (Fig.
3). Rhagoletis emerge from their
fruits as late-instar larvae and overwinter in the soil in a facultative pupal
diapause. The earlier fruiting time of apples therefore results in apple flies
having to withstand warmer temperatures for longer periods prior to winter. As
a result, natural selection favors increased diapause intensity, or greater
recalcitrance to cues that trigger premature diapause termination in apple
flies.

Figure 3. Fruit on apple trees ripens 3-4 weeks earlier than hawthorn fruit (dashed lines). Apple flies eclose earlier as adults (solid lines) and are exposed to warmer temperatures as pupae in the soil for a longer period of time before winter.

Jeff had the perfect experiment frozen in
his freezer from 20 years ago. Previously, his lab had reared the ancestral haw
race of Rhagoletis under the phenological
conditions of both host plants it attacks in nature. He had previously looked
at changes in a set of allozymes and microsatellites, but did not have the ability at the time to look across
the genome at tens of thousands of SNPs. Specifically, he exposed ancestral hawthorn
fly pupae to warm temperatures for a short 7-day (‘hawthorn-like’ control) vs.
long 32-day (‘apple-like’ experimental) period prior to winter (Fig. 4).

Figure 4. In the selection experiment, hawthorn flies were exposed to a short (7-day) versus long (32-day) prewinter period to emulate the time difference experienced by hawthorn versus apple-fly pupae in nature.

We also had a
specific hypothesis we wanted to test that integrated Jeff’s selection
experiment with sampling from natural populations. We tested whether the
changes across the genome induced by the lab experiment on divergent host-plant
phenology would predict the genome-wide differences observed at these same loci
between natural sympatric populations. In this experiment, we stressed that we
were quantifying the total genome-wide impact of selection, which involves both
direct effects, where natural selection favors the causal variants underlying
selected traits, and indirect effects, where additional loci respond because
they are correlated due to linkage disequilibrium with these causal variants. Thus,
the ‘total’ impact of divergent selection (i.e. direct + indirect effects) that
we quantify here can involve changes at many loci (Gompert et al. 2014;
Soria-Carrasco et al. 2014).

Quantifying the
impact of selection genome-wide is important because, as populations diverge,
the effects that individual genes have on reproductive isolation (RI) can
become coupled, strengthening barriers to gene flow and promoting speciation (Barton
1983, Bierne et al. 2011). If predicated solely on new mutations, this
transition could take a long time and populations could go extinct or
conditions change without speciation, which may explain why sympatric speciation
is difficult to observe and test. Thus, a prediction for systems with the
potential for speciation-with-gene-flow is that they exhibit large stores of
standing variation and consequently, show extensive, genome-wide responses to
selection when challenged by divergent ecology.

In our selection
experiment, about 6% of the SNPs showed significant frequency shifts between
the short and long prewinter periods. However, because of extensive linkage
disequilibrium (LD) in Rhagoletis,
these SNPs did not provide an estimate of the independent number of gene
regions influenced by selection. Thus, we assessed the pattern of LD between
SNPs to delimit independent sets of loci.We determined that the 6% of responding SNPs represented 162 different
sets whose members were in LD with each other, but in equilibrium with all
other SNPs. After accounting for the table-wide null expectation of 52
significant sets due to type I error, using a modeling approach we detail in our
Supplemental material, a lower bound estimate of 110 gene regions responded to
selection. To determine how physically widespread the response was across the
genome, we constructed a recombination linkage map for Rhagoletis that contained 2,352 SNPs. About 13% of mapped SNPs
showed significant frequency shifts in the selection experiment and were
dispersed widely across the five major chromosomes of the R. pomonella genome (Fig. 5). Thus, numerous independent gene
regions responded to selection and they were distributed throughout the genome.

Now we tested
our main hypothesis: does the genomic response in the selection experiment
reflect nature?The answer is yes. The
direction and magnitude of allele frequency changes for all 32,455 SNPs in the
selection experiment was highly predictive of genetic differences between the
sympatric hawthorn and apple host races at the Grant, MI, site (r = 0.39, P <
10-6). Most strikingly, for the SNPs showing significant responses in
both our selection experiment and host divergence in nature, the allele that
increased in frequency in the hawthorn race after selection was the exact same
allele in higher frequency in the apple race in nature (P = (½)154 =
4.4x10-47).

To what extent
did the single bout of selection on hawthorn flies genetically create the
derived apple race?The answer is a good
deal. For all 32,455 SNPs, the mean SNP frequency for hawthorn flies surviving
the long prewinter treatment shifted 38.9% of the difference between the host races
toward apple flies. For the 154 SNPs showing significant responses in
the selection experiment and host divergence, the shift was 84.1%.

Why is the
impact of divergent ecological adaptation so pronounced and pervasive in Rhagoletis?One contributing factor is the extensive LD
in the fly, some of which is due to inversions, requiring additional DNA
sequence analysis to resolve. A second factor is the presence of substantial
standing genetic variation in R.
pomonella, which supports the hypothesis that such stores may define taxa
having a greater capacity for speciation-with-gene-flow. Finally, when
ecological adaptation involves traits like diapause that can be highly
polygenic, selection may more often have genome-wide consequences. In this
regard, microarray studies of R.
pomonella have revealed hundreds of loci varying in expression during
diapause breakage that are potential targets of selection(Ragland
et al. 2011).

Interestingly,
this work shares some important similarities and differences with other recent
studies combining selection experiments with surveys of genome-wide genetic
variation in natural populations, including the Timema ecotypes that are the mainstay of the Nosil lab. In both a
within-generation (Gompert et al. 2014; similar to the Rhagoletis study here) and a between-generation study of selection
in the field (Soria-Carrasco et al. 2014), a genome-wide response involving
many loci was observed. However, LD was much lower in the Timema ecotypes, and thus the association between genetic differences
induced in those selection experiments did not match natural genetic variation
as closely as in the Rhagoletis
experiment.

In summary,
divergent ecological selection can have genome-wide effects even at early
stages of speciation. Large stores of standing variation in Rhagoletis fliesmay
potentiate the evolution of genome-wide reproductive isolation and their
adaptive radiation with gene flow. As the study of speciation genomics expands,
it will be possible to test the degree to which other taxa prone to ecological
sympatric speciationshare similar characteristics as R. pomonella, and to assess the relationship
between standing variation and clade richness.

Monday, June 15, 2015

I just returned last night (37 hours in transit!) from my
first trip to Uganda. It was my second trip to Africa, with the first being to
South Africa six years ago. The main purpose of the trip was to plan, with my
colleague Lauren Chapman,
some new studies on adaptation by fishes to extreme (low oxygen) environments.
However, my first trip to any new location also becomes an adventure in natural
history and photography. During these adventures, I was motivated to write a
post based on a series of natural history anecdotes, which I will also seek to
tie into a book I happened to reading at the same time.

Following in Lauren's foot steps.

My current bed time (and plane time) reading is The
Monkey’s Voyage by Alan de Queiroz. The subtitle of the book is How Improbable Journeys Shaped the History
of Life. The goal of the book is to contrast old and new views of
biogeography, the study of where species are found and why. The old view is
that the distribution of organisms and faunas across the world is almost
entirely shaped by vicariance events that sunder formerly contiguous
landmasses. These events including land masses splitting through continental
drift, mountain ranges rising, large rivers forming, and so on. Under this view,
the species found in New Zealand, for example, are remnants of an early
Gondwanaland biota that persisted (and diversified) following the isolation of
New Zealand from a larger land mass that included Australia. By contrast, the
new view is that the distribution of terrestrial organisms is shaped to a larger
extent by rare long distance dispersal across even large ocean distances. Under
this alternative view, New Zealand’s fauna is mainly shaped by over-water
dispersal from Australia long after the two islands split apart.
(Interestingly, the new view is actually an even older view. Darwin spent
considerable time studying mechanisms of long distance dispersal, although
perhaps he wouldn’t have if continental drift had been known.) De Queiroz clearly
favors the new view, marshalling extensive evidence that biogeography is
strongly shaped by long distance dispersal.

Reading biogeography in books is interesting but
experiencing it in person is transformative. For the last 14 years, most of my field
work has taken place in South America, including Trinidad, Galapagos, Panama,
and Chile, alongside shorter trips to Brazil, Barbados, Roatan, and other
locations. Now my recent trip to Uganda, combined with my earlier South African
trip, has brought home in a personal sense the differences between “New World”
and “Old World” biotas.

A South African lion showing off his dental array.

A South African hippo showing off his even more impressive dental array.

The most in-your-face contrast, of course, would be the classic African large-mammal spectacles: elephants, hippos, buffalo, giraffes, lions, leopards, wildebeest, zebras, cheetahs, camels, and so on – most of which I have now seen in the wild. Although the New World certainly does have large mammals (moose, bison, bears, capybaras, tapirs, jaguars), they are not nearly as striking, abundant, or dramatic a spectacle. However, this contrast is somewhat disingenuous given that the New World had many similar forms (mastodons, mammoths, lions, sabre-toothed cats, camels) until their extinction in the Pleistocene not that long ago. (And, of course, bison recently did, and caribou still do, present huge migratory spectacles.) So, but for vagaries of our particular point in time, the large-mammal faunas of the two continents might not have seemed quite so different.

A brown bear from my cabin in Northern BC, Canada.

Yes, moose are huge - this one in Lake Nerka, Wood River Lakes, Alaska.

A classic contemporary contrast is Old World
monkeys (and apes) versus New World monkeys. The two groups differ in a number
of ways, including various aspects of facial shape and – iconically – the
prehensile tail of New World but not Old World monkeys. In Panama, I have been
able to observe howler monkeys, white-faced monkeys, spider monkeys, Geoffroy’s
tamarins, and others. In Kibale National Park in Uganda, I was able to observe olive
baboons, grey-cheeked mangabeys, blue monkeys, redtail monkeys, red colobus,
black-and-white colobus, L’Hoest’s monkey, galagos (bush babies), pottos, and –
the most amazing of all – chimps. (Kibale is said to harbor the highest primate
biomass in the world.) Later at Lake Nabugabo, I saw vervets (more about these
later), which I had also – along with baboons – seen in South Africa. Excepting
chimps and baboons, and despite some differences in appearance, the two sets of
monkeys strike one as superficially similar. They all move with varying degrees
of frenetic activity through forest canopy feeding on a diversity of insects,
leaves, and fruits. Thus, we here have a similar ecological set of organisms in
the two worlds, with the new world monkeys having radiated from a single common
ancestor colonizing the new world, perhaps by long-distance dispersal of just a
few individuals from Africa (as argued by de Queiroz and others).

For birds, the classic contrast is between hummingbirds and
sunbirds. Hummingbirds, such a ubiquitous, striking, and engaging component of New
World environments, are entirely absent from the Old World. Instead, the Old
World has a large radiation of the nectar-feeding sunbirds. The first sunbird I
ever saw was in Cape Town, South Africa. I was on Table Mountain composing a
photograph of a flowering bush in the foreground with Cape Town in the
background far below. In the midst of a sequence of photographs, a sunbird
landed right in the middle of the flowers – almost as if I had planned for it. In
Kibale and Queen Elizabeth Parks in Uganda, I saw more sunbirds. However,
despite the similar ecologies and exuberant colouration of both groups, which
are not closely related, no one would mistake one for the other. For instance,
the hovering flight of hummingbirds – perhaps their most obvious feature – is relatively
rare in sunbirds.

Bronze sunbird, Kibale National Park, Uganda.

Both continents have wonderful radiations of small colorful frogs.
While traveling along the swampy edge of Lake Nabugabo, we had stopped so I could
take pictures of birds when one of the field assistants pointed to a tiny
yellow-and-black-patterned frog on a reed we were holding on to. Mediocre bird
photos immediately forgotten, I switched to macro equipment and started taking
endless photographs of the frog. Then, in the space of just a few minutes, he
pointed out two other species of frog clinging to other reeds less than a meter
away. All were colorful but in various shades and patterns of green. Moreover,
they all froze in place and didn’t move no matter how close my hands got or how
much I manipulated the reeds or stuck a macro lens in their faces. This appearance
and behavior was a surprise after the poison-dart frogs that I had seen in
Panama and elsewhere in the Americas. When I asked the field assistants if these
frogs were poisonous, they did not think so. It seems this group has
specialized on camouflage whereas the New World Dendrobatids have specialized
on conspicuousness. (I am no frog expert – perhaps a radiation of poisonous and
conspicuous frogs exists in Africa – and I am generalizing – some New World
frogs are very cryptic.)

Lake Nabugabo frog #1, which I still haven't taken the time to identify to species.

Lake Nabugabo frog #2, which I still haven't taken the time to identify to species.

Lake Nabugabo frog #3, which I still haven't taken the time to identify to species.

We later visited another part of the swamp and the same field assistant found another small-and-green-themed frog of seemingly yet another species. At this point, I was starting to feel incompetent in my ability to find critters and decided that I would find my own damn frog. So I went walking slowly through the marsh scanning blades of grass and other vegetation. Half an hour later, having still had no luck, I was about to give up when I saw a bit of movement near the water. “YES” I yelled, “I finally found one of the buggers” and, then, looking closer, I saw it was actually a finger-length chameleon. Even better – my first ever chameleon; and I found it myself (considerable boasting followed). The next hour was spent taking 217 photographs of the chameleon plus additional shots of what appeared to be a fifth small-green frog species that another field assistant found. Chameleons are another major radiation in the Old World – especially Madagascar – that is completely absent from the New World, which instead has a radiation of Anolislizards that are absent from the Old World.

My chameleon. Found at Lake Nabugabo, Uganda.

Getting a closer look at my chameleon. Found at Lake Nabugabo, Uganda.

Lake Nabugabo frog #4, which I still haven't taken the time to identify to species.

Lake Nabugabo frog #5, which I still haven't taken the time to identify to species.

To
these Old versus New faunal contrasts that I already knew, the present trip
added another. After having seen and photographed most of the diurnal primates,
Lauren took me on a night walk to look for the nocturnal primates. Almost
immediately, we saw a potto, which I am told is not common, and I was able to
get some photographs with a long lens and flash. What immediately struck me about
the potto was its slow, branch-hugging movement; kind of like a sloth. Hmmmm, what
about sloths? Sure enough, Lauren confirmed that sloths are absent from the Old
World just as potto-equivalent primates are essentially absent from the New
World (although the latter does have night monkeys).

Potto doing its sloth imitation in Kibale National Park, Uganda.

In this post, I have been a Natural History Tourist, giving
my own superficial impressions of some differences between the two “worlds.” Although
these impressions are based on relatively little experience in Africa, they made
me ponder the biogeography debate I had been reading in de Queiroz’s The Monkey’s Voyage. Long distance over-water
dispersal has certainly shaped the world’s fauna but vicariance ultimately
seems more important through its role in limiting movement between land masses.
On the one hand, long-distance dispersal does happen and is critical in shaping
species distributions: without it we would not have any organisms on oceanic
islands and many iconic organisms of large islands and continents would also be
missing, including perhaps monkeys in the New World. On the other hand, faunas
differ so much from place to place that effective long-distance dispersal must
be very rare and vicariance provides the dominant factor assembling many
communities.

I doubt de Queiroz would disagree with these points even
though his book is very much focused on long-distance dispersal. Another way to
explain the distinction is that long-distance dispersal is critical for explaining why species ARE in particular places whereas vicariance is critical for explaining why species ARE NOT in particular places.

All of this brings me back, believe it or not, to vervets,
the first monkey I ever saw in the wild. This statement might seem surprising
if you remember that vervets are Old World Monkeys whereas I had worked in
South America for eight years before visiting Africa. In fact, my first
experience with wild monkeys – the vervets – was in 2003 in Barbados. Yes: Old
World Monkeys on New World Islands! It turns out that vervets were brought about
350 years ago to Barabados and some other Carribean islands by slavers. I don’t
think other monkeys are naturally found on those islands, at least not on
Barabados, so this isn’t a lesson in what happens when the two faunas collide. But
if they did collide, who would win? Are New World Monkeys “better” than Old
World monkeys? (They have that cool tail!) Would New World monkeys win in the
New World and Old World monkeys win in the Old World (local adaptation!) – or vice
versa (enemy release!)? Of course, countless such experiments are being undertaken
with other organisms, as the field of invasion biology attests, but I don’t know
any examples of recent human-mediated conflicts between ecologically-equivalent
iconically-divergent faunas such as those described above. (Although placental
dingos replaced, whether causally or not, the marsupial thylacine in
Australia).

A Barbados vervet

How interesting it would be to bring
hummingbirds to the Old World and sunbirds to the New, Anolis lizards to the Old and chameleons to the New, capybaras to
the Old and hippos to the New, and so on. (Apparently Pablo Escobar, the drug
lord, had a hippo herd that is now feral and expanding in
Columbia.) Just think how much we would learn and how fun it would be to
see the dynamics play out. Sadly, however, the likely ecological impact would
exceed the value of the information gained thereby. I would much rather see Anolis in South American and chameleons
in Africa than I would like to find out who would win in direct competition in
nature. It is too bad we can’t have replicate worlds, some for conservation and
some for experimentation.

Wednesday, June 3, 2015

When thinking
about ecological speciation, we often assume that habitat-related local
adaptation drives genetically based population divergence, and that the
resulting phenotypic divergence promotes reproductive
isolation. However, strong phenotypic divergence and associated
reproductive isolation might also arise directly via phenotypic
plasticity. In a paper that just went online, we start exploring the
latter pathway to speciation in lake and stream stickleback
populations in the Lake Constance basin (Central Europe). This system
is cool because the stream populations consistently show the relatively
small body size at reproduction typical of stream stickleback worldwide
(Moser et a. 2012, PLoS One), whereas the
lake fish reproduce at much larger body size (the photo below shows a representative reproductive male from Lake Constance and a tributary). Given that experimental
work in many stickleback systems has shown that body size differences
between populations represent a major sexual reproductive barrier, we
wanted to understand how the body size differences
among lake and stream fish arise. A combination of laboratory and field
transplant work (see photos below) revealed that the divergence seen in the wild is
largely plastic, most likely driven by differences in resource
availability between the two habitat types: lake fish use
a relatively poor limnetic food base, grow slowly, fail to reach
reproductive size after one year, and hence reproduce only after two
years at large size. Stream fish, by contrast, exploit a rich benthic
food base, reach critical size after one year, reproduce
directly at relatively small size, and die. In the laboratory and in
field transplants, these life history differences disappear. We thus
hypothesize that in this system, sexual reproductive barriers might have
established immediately due to plastic divergence
in life history. Evaluating this hypothesis experimentally is the next
step. Also, it would be great to know if in other lake-stream
stickleback systems, body size differences do have a stronger genetic
component than in the Lake Constance basin.